Direct cholesterol assay reagent

ABSTRACT

The present invention relates to the direct quantitative determination of cholesterol and involves the formation of a spectrophotometrically active product of cholesterol obtained by contacting cholesterol with an acyl compound and a perchlorate effective to form the spectrophotometrically active product.

This application is a continuation of U.S. Ser. No. 08/432,653, filedMay 2, 1995, now abandoned, which is a divisional of U.S. Ser. No.08/091,499, filed Jul. 14, 1993, now abandoned, which is a Continuationin Part of U.S. Ser. No. 07/785,998, filed Oct. 31, 1991, now U.S. Pat.No. 5,252,488, which is a Continuation-in-Part of U.S. Ser. No.07/639,222, filed Jan. 9, 1991, now U.S. Pat. No. 5,246,864, which is aContinuation-in-Part of U.S. Ser. No. 07/463,473, filed Jan. 11, 1990,now abandoned.

FIELD OF THE INVENTION

The present invention is related to a method of forming aspectrophotometrically active cholesterol product that is particularlyutile in clinical detection methods for cholesterol using, for example,fluorescence spectrophotometry, derivative absorption spectrophotometry,circular dichroism and more especially absorption spectrophotometry.More specifically, the present invention permits the direct,simultaneous quantitative determination of total cholesterol andcholesterol subfractions in clinical samples. The present invention alsorelates to a chemical reagent system useful for formingspectrophotometrically active products with analytes such as cholesteroland to a spectrophotometer apparatus useful in the aforesaid absorptiondetection chemical methods.

BACKGROUND OF THE INVENTION

Although cholesterol levels are widely regarded as a reliable indicatorof prospective health problems attributable, for example, to coronarydisease, these levels are not easily measured by routine analyticalmethods involving spectrophotometry since cholesterol is not a coloredmaterial. Thus previously known methods for cholesterol determinationgenerally require a color derivatization step. Examples of these includethe Liebermann-Burchard reaction, the Zak reaction and the Abell-Kendallreaction, all of which occur in non-aqueous media and require specialcontrol of reaction conditions. Another reaction known in this regard isthe oxidizing enzyme-dye reaction.

The color derivatization steps known heretofore, however, do not providea derivative of the cholesterol molecule itself; rather, they providesecondary products of cholesterol oxidation which means that thespectrophotometric measurement of cholesterol is an indirect one. Thatis, the intensity of the color is not a direct measure of cholesterolconcentration.

As noted hereinbefore, most routine analytical methods for practicalpurposes employ spectrophotometry. Spectrophotometry refers to themeasurement of the absorption or transmission of incident light throughsolutions of test compounds. Typically, compounds of interest havecharacteristic spectra, transmitting or absorbing specific wavelengthsof light, which can be used to determine the presence of these compoundsor measure their concentration in test samples. Instruments designed forspectrophotometric absorption have a light source, for which the emittedwavelength is known and may be adjusted, and one or more detectorssensitive to desired wavelengths of transmitted or reflected light.Spectrophotometric absorption can be used to determine the amount of agiven compound that is present in a test sample.

Circular dichroism (CD) is a special type of absorption method in whichthe molecular composition of an analyte results in differentialabsorption of incident light not only at a specific wavelength but alsoof a particular polarization state. Circular dichroism is a chiropticalmethod which allows one to differentiate between different enantiomers;that is, optical isomers having one or more asymmetric carbon atom(chiral) centers. When utilizing CD, generally a sample is illuminatedby two circularly polarized beams of light traveling in unison. Bothbeams pass through the sample simultaneously and are absorbed. If thesample is optically active, the beams are absorbed to different extents.The differences in absorption of the beams can then be displayed as afunction of the wavelength of the incident light beam as a CD spectrum.No difference in absorption is observed for optically inactive absorbersso that these compounds are not detected by a CD detecting system. Theuse of CD as a chiroptical method has been fully described in scientificliterature, such as Lambert, J. B. et al. "Organic Structural Analysis",Macmillan, New York, N.Y. 1976.

Early applications of the CD method dealt primarily dealt withelucidation of molecular structures, especially natural products forwhich a technique capable of confirming or establishing absolutestereochemistry was critical. However, CD has also reportedly been usedin a clinical method to quantitatively determine unconjugated bilirubinin blood plasma, Grahnen, A. et al. Clinica Chimica Acta, 52, 187-196(1974). In the method thus disclosed, a complex was formed betweenbilirubin and human serum albumin as a CD probe for bilirubin analysis.

Clinical applications of circular dichroism are also discussed by NeilPurdie and Kathy A. Swallows in Analytical Chemistry, Vol. 61, No. 2,pp. 77A-89A (1989), herein incorporated by reference. Possible clinicalapplications of CD are disclosed to include measurement of cholesterollevels and detection of anabolic steroids. However, suitable chemicalreagents for carrying out such testing are not disclosed.

Regarding the use of spectrophotometric absorption, fluorescence,derivative spectrophotometry or CD methods herein disclosed to measurecholesterol levels, it is noted that the population at large iscontinually advised that it is prudent to know serum cholesterol levelsand constantly reminded that an uncontrolled diet and a lack of exercisecan lead to accumulation of arterial plaque that will increase the riskof atherosclerosis and coronary heart disease. Statistical studies, suchas those reported by Kannel, W. B. et al. in "Serum Cholesterol,Lipoproteins and the Risk of Coronary Heart Disease: The FraminghamStudy" Ann. Intern. Med., 74:1-11 (1971) and Castelli, W. P. et al. in"Incidence of Coronary Heart Disease and Lipoprotein CholesterolLevels", JAMA, 256:2835-2838 (1986), have shown that other risk factors,such as age, gender, heredity, tobacco, alcohol consumption etc. mustalso be considered when counselling patients about the risks.

The magnitude of the program for screening the general public is soimmense that automated methods for cholesterol determinations arenecessary. The tests currently used differ in complexity from the simpledip-stick approach, which uses a color sensitive reaction on a papersupport, to sophisticated lipid profile tests in which the distributionof cholesterol among the various solubilizing molecular species isdetermined, Abbott, R. D. et al. "Joint Distribution of LipoproteinCholesterol Classes, The Framingham Study, Arteriosclerosis, 3:260-272(1983). Here, the dip-stick approach is only a preliminary qualitativetest upon which a decision for the fuller, more quantitative measurementcan be based.

At the conclusion of a recent extensive study of how health risk factorsare related to elevated levels of serum cholesterol, a report entitled"Current Status of Blood Cholesterol Measurement in ClinicalLaboratories of the United States, A Report from the LaboratoryStandardization Panel of the National Cholesterol Education Program",Clin. Chem., 34:193-201 (1988), was prepared by the LaboratoryStandardization Panel (LSP) of the National Cholesterol EducationProgram (NCEP). In this report, the measure or risk was correlated withthree ranges of total cholesterol (TC): low risk if less than 200 mg/dL;moderate risk in the range 200-239 mg/dL; and high risk if greater than240 mg/dL. In order to place a particular individual into one or anotherof these categories, all that is required is a serum TC measurement. Theother risk factors, such as those identified by Kannel et al. andCastelli et al., supra, are then added as a basis for further patientcounselling. This relatively simple approach replaces an earlierrecommendation in Kannel et al., supra, and in Superko, H. R. et al."High-Density Lipoprotein Cholesterol Measurements--A Help or Hinderancein Practical Clinical Medicine", JAMA 256:2714-2717 (1986) in whichrelative risk was established using a ratio of TC to high densitylipoprotein cholesterol (HDL-C) equal to 5. A ratio lower than 5 impliesa high level of HDL-C and a low relative risk. For this diagnosis, HDL-Cis measured in a second independent test.

The same report by the LSP hastened to add that there were seriousinaccuracies in measurements made by numerous clinical laboratories inthe determination of the amount of TC present in human serum referencestandards.

Statistically the results showed that, in data from 1500 laboratories,47% failed to measure the true value to within a coefficient of variance(CV) of ±5%, and 18% of these failed at a CV of ±10%. As a consequence,the LSP recommended that an improvement in CV to within ±3% for TCshould be achieved by 1992. Recent surveys indicate that certifiedlaboratories are well on their way to meeting that challenge, using thecurrent clinical methods and instrumentation, as reported, e.g. inPosnick, L. "Labs now Better at Cholesterol Tests Data Show", Clin.Chem. News 15(9):14 (1989). The LSP did not report the inaccuraciesassociated with the determination of the distribution of cholesterolamong the various lipids and lipoproteins, but did indicate that anevaluation would be made in the future. The very poor proficiency andlack of reliability in the measurement of serum or plasma HDL-C, hasbeen eloquently described in Superko, H. R. et al., supra, and inWarnick, G. R. et al. "HDL Cholesterol: Results of InterlaboratoryProficiency Test" Clin. Chem. 26:169-170 (1980) and Grundy, S. M. et al."The Place of HDL in Cholesterol Management. A Perspective from theNational Cholesterol Education Program" Arch. Inter. Med. 149:505-510(1989) where interlaboratory CV's as high as 38% were reported. A 1987evaluation by the College of American Pathologists (CAP) of themeasurement of the same sample for HDL-C by over two thousandlaboratories showed that more than one third differed by more than 5%from the reference value. Interlaboratory CV's amount groups using thesame method did improve to 16.5%, but it is still too imprecise to be ofany predictive clinical value. For this reason, the TC:HDL-C ratio is nolonger used in risk assessment, although it offers potential advantagesin defining the true clinical picture.

Regarding presently used lipid profile studies, cholesterol is known tobe distributed in the serum mainly associated with high densitylipoprotein (HDL-C) and low density lipoprotein (LDL-C) fractions andwith triglycerides as the very low density lipoprotein cholesterol(VLDL-C) fraction. There is plenty of statistical evidence from a numberof long term clinical tests to indicate that a high proportion of HDL-Cand a low proportion of LDL-C is associated with lower relative risk orin simpler terms, high levels of LDL-C are to be avoided where possible.HDL-C is beneficial, provided the level is not excessively low, i.e.,less than 30 mg/dL. VLDL-C cholesterol has not been implicated in anyrisk determination, but high triglyceride itself can be a seriousproblem.

In a typical lipid profile study, total cholesterols are measureddirectly and HDL-C is measured in the supernatant remaining aftertreatment of the sample with an agent to precipitate out LDL-C andVLDL-C. VLDL-C is taken to be a fixed fraction (e.g., 0.2) of thetriglyceride, which is also measured directly in a separate assay. LDL-Cis calculated from these figures and is not measured directly. Thepropagation of errors in each of the three independent measurements makeLDL-C the fraction known with least overall accuracy and precision,although it may be the most significant aspect of cardiovascular risk.Because of this, it is difficult to meaningfully monitor and establishthat clinical progress has been made in LDL-C reduction therapy withtime.

At a workshop and subsequent roundtable session held at the 43rd Meetingof the American Association for Clinical Chemistry, the present state ofthe art in this area was summarized, as reported in Baillie, E. G. etal. "Standardization and Clinical Utility of Lipid Determinations",Workshop, 43rd National Meeting American Association for ClinicalChemistry, 1991 and Warnick, G. R. "Standardization of HDL CholesterolMeasurement" Roundtable, 43rd National Meeting, American Association forClinical Chemistry, 1991. From these proceedings, it was concluded thataccuracy is essential in HDL-C measurement. While presently availableprecipitation methods can give satisfactory results, the values obtainedby these methods in routine clinical laboratory settings do not meetreal medical needs. The CAP comprehensive chemistry proficiency surveyfrom 1982 to 1991 for HDL-C showed interlaboratory CVs of about 20% in1991, with no overall improvement since 1982. The CVs delivered byclinical instruments used for HDL-C measurements ranged from 7.6% forthe Dimension to 50% for the Ektachem.

At the sessions, it was also noted that direct methods for LDLcholesterol are needed. The use of triglyceride determinations toestimate VLDL-C by the Friedewald equation is, at present, the method ofchoice. To quote the workshop syllabus, "The variability typicallyobserved in the measurement of total and HDL cholesterol andtriglycerides may preclude attaining acceptable precision. In fact, toachieve the ideal precision in LDL cholesterol estimation, the precisionof the constituent measurements must be better than their idealspecifications."

It is thus established that there is a need for a relatively simple,reliable and repeatable assay method to directly and simultaneouslydetermine the amount of cholesterol, both total cholesterol and itsdistribution among the various subfractions without the need forprecipitation or separate measurements of these subfractions.

SUMMARY OF THE INVENTION

The present invention provides a method of forming aspectrophotometrically active cholesterol product which can be employedin an assay for the direct, simultaneous, quantitative determination oftotal cholesterol and its distribution among HDL-C, LDL-C and VLDL-Csubfractions. The spectrophotometric activity of the product may bemeasured by conventional absorption, fluorescence, first and secondderivative spectrophotometrics, as well as circular dichroism.

Thus, in one embodiment the present invention provides a method forforming a spectrophotometrically active product of cholesterol whichcomprises contacting cholesterol with an acyl compound and a perchlorateeffective to form a spectrophotometrically active product with thecholesterol. When formed in a test sample, for example, thespectrophotometric activity may be evaluated to determine the amount ofcholesterol present in the sample, including its distribution amongHDL-C, LDL-C and VLDL-C subfractions. The method of this embodimentpermits a quick and repeatable method for the direct, simultaneousquantitative determination of cholesterol, both total cholesterol andsubfraction distribution, at ambient temperature, without the need forprecipitation or separate subfraction measurement.

In another embodiment the present invention provides for a clinicalmethod for determining the amount of cholesterol (in cholesterolsubfractions) in a test sample, by forming a reaction product with thecholesterol and then either performing step (a¹), (a²), (a³) or (a⁴),wherein steps (a³) and (a⁴) may be followed by calculation ofcholesterol concentrations using matrix mathematics and constantsderived for the particular cholesterol subfraction analyzed:

Step (a¹) determining the CD absorption spectrum of the test sample overa range from about 150 to 700 nm (preferably from about 360 nm to 700nm);

Step (a²) determining the CD absorption of the test sample at one ormore discrete wavelengths within a range from about 150 nm to 700 nm(preferably from about 360 nm to 700 nm);

Step (a³) determining the spectrophotometric, fluorescence or derivativespectrophotometric absorption of the test sample at three or morediscrete wavelengths within a range from about 150 nm to 700 nm(preferably about 360 nm to 700 nm);

Step (a⁴) determining the spectrophotometric fluorescence or derivativespectrophotometric absorption of the test sample over a range from about150 nm to 700 nm (preferably from about 360 nm to 700 nm).

The invention further provides novel absorption detection apparatusesfor practicing certain of the present inventive methods, whichapparatuses are exemplified, but not limited, by the following:

A spectrophotometric absorption instrument for determining the amount ofVLDL-C, LDL-C, HDL-C and TC present in a test sample, the instrumentcomprising means for determining the spectrophotometric absorptionspectrum of the test sample at 3 or more distinct wavelengths, withinthe range of about 150 to 700 nm (preferably 360 nm-700 nm), and meansfor determining the amount of VLDL-C, LDL-C, HDL-C and TC present in thetest sample based on, for example, the spectrophotometric absorption ofthe cholesterol reaction products in the test sample. Optionally, theinstrument further comprises means for adding reagent to the testsample.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given here and below and the accompanying drawingswhich are given by way of illustration only, and thus, are notlimitative of the present invention.

FIG. 1 is a full CD spectrum for the optically active colored productobtained from the reaction of Chugaev reagents with cholesterol. Curve(a) is representative of the total cholesterol, while the shaded area isthe spectrum after the addition of the LDL-C, VLDL-C precipitating agentand is therefore representative of the HDL-C fraction only.

FIG. 2 represents the correlation between TC as measured in serumsamples processed by two different labs using prior art processes (LabsA and B), versus total cholesterol as measured by the CD method of thepresent invention (this lab); y=-10.209+1.0055x, R² =0.835.

FIG. 3(a) is a graph of TC vs. (VLDL+LDL)-C using a CD method of thepresent invention (this lab); y =5.0554+0.84693x, R² =0.932.

FIG. 3(b) is a graph of TC vs. (VLDL+LDL)-C using a prior art process(LAB-A); y=-47.672+0.98751x, R² =0.987.

FIG. 3(c) is a graph of TC vs. (VLDL+LDL)-C using a prior art process(LAB-B); y =046.5222+0.9869x, R² =0.98.

FIG. 4(a) is a graph of TC vs. HDL-C using the CD method of the presentinvention (this lab); y -5.2861+0.14995x, R² =0.335.

FIG. 4(b) is a graph of TC vs. HDL-C using a prior art process (LAB-A) ;y=47.648+0.012569x, R² =0.001.

FIG. 4(c) is a graph of TC vs. HDL-C using a prior art process (LAB-B);y=46.522+0.0131x, R² =0.06.

FIG. 5 is a schematic of a CD, wherein:

LS is the high intensity conventional light source or laser source; M1and M2 are monochromators required for full spectral data; P is thelinearly polarizing element; Q is the circularly polarizing element; Sis the sample cell; D is the detector (of which there may be up tothree); and REC is the recorder.

FIG. 6 is a graph of the absorption spectrum of whole serum over thewavelength range of 400 nm-700 nm.

FIG. 7(a) is a graph of the fluorescence spectrum for VLDC-Csubfraction;

FIG. 7(b) is a graph of the fluorescence spectrum for LDL-C subfraction;

FIG. 7(c) is a graph of the fluorescence spectrum of HDL-C subfraction;

FIG. 8(a) is a graph of the first (solid line) and second (dotted line)derivative of the conventional absorbance spectrum of the VLDL-Csubfraction (sigma);

FIG. 8(b) is a graph of the first (solid line) and second (dotted line)derivatives of the conventional absorbance spectrum of the LDL-Csubfraction (sigma);

FIG. 8(c) is a graph of the first (solid line) and second (dotted line)derivatives of the conventional absorbance spectrum of the HDL-Csubfraction (sigma);

FIG. 9(a) is a graph of the first (solid line) and second (dotted line)derivatives of the Serum A test sample; and

FIG. 9(b) is a graph of the first (solid line) and second (dotted line)derivatives of a Serum B test sample.

FIG. 10 is a graph of the absorbance spectra for three samples whereintotal cholesterol for each sample was similar but triglyceride (TG)levels varied from about 30 mg/dL (Curve a), about 306 md/dL (Curve b)and about 630 mg/dL (Curve c).

FIGS. 11a, 11b and 11c are graphs respectively depicting the correlationbetween total cholesterol (TC), VLDL-C and LDL-C in 35 test samples asmeasured by the spectrophotometric active product in accordance with thepresent invention (denoted as "spec") and as measured by a commerciallyavailable technique (denoted "enzym").

FIGS. 12a and 12b are graphs depicting the correlation between HDL in 35test samples as measured by the spectrophotometrically active product inaccordance with the present invention (denoted as "spec") and asmeasured by a commercially available technique (denoted as "enzym").

FIGS. 13a, 13b and 13c are graphs depicting the correlation between TC,LDL and HDL in several hundred samples as measured by thespectrophotometrically active product in accordance with the presentinvention (denoted as "spec") and as measured by a commerciallyavailable technique (denoted as "enzym").

FIGS. 14a and 14b are graphs depicting the correlation between VLDL asmeasured by the spectrophotometrically active product in accordance withthe present invention (denoted as "spec") and as measured by acommercially available technique (denoted as "enzym") wherein thesamples had TG<400 mg/dL (FIG. 14a) and wherein TG was between 400 and1000 mg/dL (FIG. 14b).

FIGS. 15a and 15b are graphs depicting the inverse correlation betweenHDL and TG as measured by a commercially available technique (denoted as"enzym") (FIG. 15a) and as measured by the spectrophotometrically activeproduct in accordance with the present invention (denoted as "spec")(FIG. 15b).

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the present invention is providedas an aid in the practice of the present invention. Much of thediscussion appearing herein relates to methods and instruments fordetermining TC and the amount of cholesterol subfractions present in atest sample, however, the present invention should not be considered tobe unduly limited by such discussions. This is true, since those skilledin the art will generally understand that the reagents, reagent ratios,reaction conditions and apparatuses herein disclosed may be modifiedwithout departing from the spirit or scope of the present invention.

The following discussion first provides a glossary of certain terms usedherein, and then considers the inventive methods herein disclosed andconcludes with a discussion of novel apparatus, which are particularlyuseful in performing the methods herein disclosed.

The following Glossary of Terms is provided to remove any ambiguity,which may exist as to the use of certain terms and abbreviations usedherein.

The term "CD instrument" as used herein, means a Circular DichroismInstrument. Such instruments are available commercially or may beconstructed from parts, which may be commercially available.Additionally, FIG. 5 is included herewith to provide a simple schematicof how a CD works. As can be seen in FIG. 5, light from a light source(LS) is linearly polarized with linear polarizers (P) and thencircularly polarized in opposite directions by circular polarizers (Q)and then shown through a specimen cell (S), whereupon absorbance ismeasured by a detector (D), the difference in absorption of theoppositely polarized light beams is measured and plotted as a functionof wavelength to produce a CD spectrum, or alternatively, may berecorded at preselected wavelengths.

The term "LDL cholesterol" (abbreviated LDL-C) as used herein, means lowdensity lipoprotein cholesterol. The term "HDL cholesterol" (abbreviatedHDL-C) as used herein, means high density lipoprotein cholesterol. Theterm "VLDL cholesterol" (abbreviated VLDL-C) as used herein, means verylow density lipoprotein cholesterol, the abbreviation "(VLDL+LDL)-C" asused herein means the combined VLDL-C and LDL-C fractions and the term"total cholesterol" (abbreviated TC) as used herein, means the sum ofthe cholesterol subfractions in a test sample, i.e.,TC=HDL-C+LDL-C+VLDL-C. The term "cholesterol subfraction" as usedherein, refers to any or all of the HDL-C, LDL-C and VLDL-C.

The term "Chugaev reagent" as used herein, means a reagent described byCox and Spencer in Can. J. Chem., 29, 217 (1951) or to reagents derivedfrom that basic reagent configuration by varying the proportions of theacetyl chloride, zinc chloride and acetic acid, or by substituting zincacetate in acetyl chloride for zinc chloride/acetic acid.

The term "Chugaev reaction product" as used herein, means any of thereaction product(s) of cholesterol with Chugaev reagents. A "Chugaevreaction" utilized herein to form a Chugaev reaction product of thepresent invention, is discussed in the above-mentioned literature of Coyand Spencer and is thought to involve dehydration and opening of theB-ring of the steroid to form an optically active colored reactionproduct.

The term "test sample", "clinical test sample" or "serum test sample" asused herein, refers to a whole blood test sample or a whole blood testsample having the cell bodies removed therefrom by means which are wellknown to those skilled in the art (e.g., by centrifugal force, afiltering mechanism or the like).

The term "spectrophotometric absorption" as used herein refers tomeasurement of the absorption (or, conversely, transmission) of incidentlight by colored compounds at specific wavelengths, irrespective of thestate of polarization of the light.

The term "spectrophotometric absorption detection" as used herein meansdetection and quantitation of analytes in a test sample by measuringtheir absorption of light at various wavelengths, without regard to thestate of polarization of the incident or absorbed light. Absorption inthis case is proportional to the number of molecules of analyte presentin the test sample.

The term "fluorescence spectrophotometry" as used herein means detectionand quantitation of analytes in a test sample by measuring the intensityof light emitted by the analytes at various wavelengths, following theirirradiation by incident light at different wavelengths. Fluorescence isproportional to the number of molecules of analyte in the irradiatedsample.

The term "first derivative spectrophotometry" as used herein describesthe spectrum that is obtained by calculating the rate of change ofabsorbance with wavelength plotted against wavelength. The term "secondderivative spectrophotometry" as used herein describes the spectrumobtained by calculating the rate of change of the first derivative withwavelength plotted against wavelength.

The term "spectrophotometrically active" or "spectrophotometricactivity" as used herein refers to a property or characteristic that isdetectable by spectrophotometric methods such as absorptionspectrophotometry, circular dichroism, fluorescence spectrophotometry,derivative absorption spectrophotometry and the like.

METHODS Direct Detection of Cholesterol Fractions Using CD Absorption,Spectrophotometric Absorption Detection, or Fluorescence or DerivativeAbsorption Spectrophotometric Methods

In a first embodiment, the present invention is directed to theintroduction of a color reaction described in the literature, Cox andSpencer, Can. J. Chem., 29, 217 (1951), as the Chugaev reaction.

The reagents utilized in making the Chugaev reaction are for example 20%w/v ZnCl₂ in glacial acetic acid, and 98% acetyl chloride. Thesematerials can be stored in separate containers and will remain usablefor many weeks, even when stored at about 40° C. Moreover, the degree oftheir dryness does not have to be carefully controlled. The product ofthe Chugaev reaction with cholesterol is reddish orange in color and isthought to be a conjugated triene CD-active derivative of cholesterol.The intensity of the color is a direct measure of the cholesterolconcentration. In contrast, dyes used in the known methods forcholesterol analysis are secondary products of cholesterol oxidation andare not derivatives of the cholesterol molecule itself. They are thus anindirect measure of the number of cholesterol molecules present in thetest sample.

If desired, the components of the Chugaev reagent may also be storedtogether in a ratio over the range of about a 1:1 to 4:1 ZnCl₂ /glacialacetic acid to 98% acetyl chloride, all of which gave satisfactoryreactions with cholesterol. Reagents must be kept when stored underairtight conditions in an amber glass, teflon or a similar container. Inthis regard, an extended period of stability was observed for reactantsstored together at 40° C. in amber bottles for at least 4 weeks.

It was observed that when ratios of 1:1 to 4:1 of zinc reagent to acetylchloride are utilized, voluminous precipitates can occur, which cannotalways be removed in a centrifugation step that follows the incubationperiod. While this is not a serious problem in CD detection, because thedifference in absorption of two beams is measured which effectivelycancels out the contribution from light scattering, it can be seriouswhen a single beam absorption detection method is used (e.g., absorptiondetection, fluorescence or derivative absorption spectrophotometry). Inthis respect, the invention has discovered that when the acetyl chlorideis used in an amount in excess of the zinc reagent (e.g., 20%-25% w/vZnCl₂ in glacial acetic acid) problems with precipitates are minimized.Most preferably the acetyl chloride is used in a high relative amount tothe zinc reagent. Such preferred ratios range from about 4:1 to 100:1.Alternatively, zinc acetate may be added directly to the acetylchloride, e.g., 0.95 mg zinc acetate dihydrate in 1.0 ml acetylchloride.

In a second embodiment, the present invention is directed to a methodfor forming a spectrophotometrically active product of cholesterol whichcomprises contacting cholesterol with an acyl compound and a perchlorateeffective to form a spectrophotometrically active product ofcholesterol.

Perchlorates particularly useful in this embodiment of the presentinvention are those which are effective to form a spectrophotometricallyactive product, such as a colored product, with cholesterol. Suchperchlorates include but are not limited to those which contain zincand/or barium, such as zinc perchlorate or barium perchlorate, andincluding hydrated forms of these; perchloric acid, which for purposesof convenience in the present specification is referred to as aperchlorate, may also be used, as may mixtures of the above. A preferredperchlorate is zinc perchlorate, such as zinc perchlorate hydrate; mostespecially zinc perchlorate hexahydrate.

Acyl compounds particularly useful in this embodiment of the presentinvention are those which, in conjunction with the above-definedperchlorates, are effective to form a spectrophotometrically activeproduct, such as a colored product, with cholesterol. An acyl compounduseful in this regard has the formula ##STR1## wherein R¹ is halogen,--NH₂, --OR², ##STR2## and R, R² and R³ are each independently loweralkyl, aryl, alkaryl, aralkyl or mixtures thereof.

As employed herein, the lower alkyl groups contain up to 6 carbon atomswhich may be in the normal or branched configuration, including methyl,ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl, penty, hexyland the like. Preferred lower alkyl groups contain 1 to 3 carbon atoms;methyl is especially preferred.

The aryl groups are aromatic rings containing from 6 to 14 carbon atoms.Examples of aryl groups include phenyl, α-naphthyl and β-naphthyl.

The alkaryl groups contain up to 16 carbon atoms with each alkyl groupcontaining up to 6 carbon atoms which may be in the normal or branchedconfiguration, and each aryl group containing from 6 to 10 carbon atoms.Preferably, each alkyl group contains 1 to 3 carbon atoms.

The aralkyl groups contain up to 16 carbon atoms with each aryl groupcontaining from 6 to 10 carbon atoms and each alkyl group containing upto 6 carbon atoms which may be in the normal or branched configuration.Preferably, each aryl group contains 6 carbon atoms and each alkyl groupcontains 1 to 3 carbon atoms.

The halogens include fluorine, chlorine, bromine and iodine. Thepreferred halogen is chlorine.

In a preferred practice, R¹ is halogen, R is lower alkyl, aryl ormixtures thereof. In a particularly preferred practice, R¹ is chlorineand R is methyl, resulting in acetyl chloride; or phenyl, resulting inbenzoyl chloride, which as those in the art will appreciate manifests acertain toxicity. Acetyl chloride is the most preferred acyl compound.

While the concentration of perchlorate and acyl compound may vary withinwide ranges as determinable by those of skill in the art, it ispreferred if about 0.3 to 0.7 molar perchlorate is present in about 90to 100% acyl compound; more preferably about 0.5 to 0.6 molarperchlorate in about 95 to 99% acyl compound; most preferably about 0.5molar in about 98% acyl compound. This last concentration is especiallypreferred when zinc perchlorate hexahydrate and acetyl chloride areemployed.

In one aspect, the present invention is directed to a method fordetermining the amount of cholesterol present in a test sample. Thecholesterol in the test sample is contacted with the aforedescribed acylcompound and perchlorate effective to form a spectrophotometricallyactive product with the cholesterol under conditions effective to formsaid product; the spectrophotometric activity of the product is thenevaluated to determine the amount of cholesterol present in the sample.The spectrophotometrically active product in this regard is capable ofdetection by conventional techniques, such as absorptionspectrophotometry, circular dichroism, fluorescence spectrophotometry orderivative absorption spectrophotometry.

In practicing the present invention, the direct, simultaneous,quantitative determination of total cholesterol and its distributionamong HDL-C, LDL-C and VLDL-C subfraction in a test sample may beobtained. Thus, upon forming the spectrophotometrically active productwith cholesterol in accordance with the present invention, thespectrophotometric activity of the product is measured, from whichmeasurement is calculated the amount of HDL-C, LDL-C, VLDL-L and totalcholesterol present in the test sample. Importantly, the method can beperformed at ambient conditions, e.g., at about room temperature and atabout atmospheric pressure, within a short period of time.

In one embodiment, it is preferred that the spectrophotometric activityof the product is measured by spectrophotometric absorption atwavelengths from between about 150 to about 750 nm; more preferablythese measurements are made at three or more distinct wavelengthscorresponding to the various subfractions among which the major portionof cholesterol is distributed. Although the wavelengths selected mayvary as the choices of reagents and conditions are changed, e.g., volumeof sample, volume of reagent, incubation temperature, incubation time,the actual values need not be specified since they are readilydeterminable by those of skill in the art. Generally, when, for example,zinc perchlorate hexahydrate and acetyl chloride are used, adequatewavelengths for the measurement of spectrophotometric absorption are atabout 410 nm, about 456 nm and about 518 nm.

From these measurements the amount of HDL-C, LDL-C and VLDL-C can bedirectly calculated by solving an algorithm which correlates absorptionto the amounts of HDL-C, LDL-C and VLDL-C present in the samples frommathematical relations known to the art. Thus, the concentrations ofHDL-C, LDL-C and VLDL-C may be calculated by solving an n·n matrix(where for example, n=3) consisting of three linear Beer's Law equationsgiven the sum of absorbances for each fraction as elaborated uponhereinbelow. Alternatively, the algorithm may utilize multivariateregression analysis which may comprise inter alia techniques ofprincipal component analysis, pattern recognition analysis or partialleast squares analysis, also as exemplified hereinbelow and commonlyavailable to the art. Importantly, in this aspect of the presentinvention, TC is determined by the summation of the subfractions.

In practicing the present invention, the rate at which thespectrophotometrically active product is formed may be controlled, i.e.,increased or decreased, by the addition of a modifier to the combinationof acyl compound and perchlorate as hereinbefore defined. Modifiersuseful to decrease the rate include water, glacial acetic acid,chloroform and like compounds and mixtures thereof. Generally, todecrease the rate, modifier is present at a concentration of greaterthan about 10% v/v based upon the acyl compound, such as acetylchloride.

Modifiers useful to increase the rate include HCl, HClO₄ and likecompounds and mixtures thereof. Generally, to increase the rate modifieris present at a concentration of about 1-2% v/v based upon the acylcompound, such as acetyl chloride.

Another aspect of this particular embodiment of the present invention isdirected to a chemical reagent which comprises an acyl compound and aperchlorate effective as hereinbefore defined to form aspectrophotometrically active product with an analyte such ascholesterol or like lipidic material, including, for example,lipoproteins, anabolic steroids or other steroidal products.

A. Direct Method Using CD

An advantage when using CD in the present invention is that CD allowsfor great specificity and selectivity in determining the amount of thedifferent cholesterol subfractions present in the test sample, i.e.,(VLDL-C+LDL-C) and HDL-C. However, a drawback is that the levels ofVLDL-C and LDL-C could not be directly separated using CD.

The full CD spectrum for the orange colored optically active productfrom the Chugaev reaction with cholesterol is shown in FIG. 1. Thesample is a chloroform solution of the NBS Cholesterol StandardReference Material (SRM911a). This spectrum is used as the referencestandard for all subsequent serum cholesterol measurements.

In the CD absorption spectrum, the HDL-C and the (VLDL+LDL)-C fractionsare associated with different spectral bands and can be measureddirectly from the same specimen, FIG. 1, without the need for aprecipitation step to determine HDL-C. In this regard, measurements at525 nm give results for the combined (VLDL-C+LDL-C) fractions andmeasurements at 390 nm (or preferably the algebraic sum of the negativeand positive CD absorption peaks at 390 nm and 475 nm, respectively)give results for the HDL-C fraction.

It is thought preferable to determine the algebraic sum of the CDabsorption peak heights at about 390 and 574 nm, when determining HDL-Clevels, since this method uniformly provides a lower coefficient ofvariation with respect to the values obtained for HDL-C, versus themethod wherein only the CD absorption measurement at about 390 nm isused. The decrease in variation with the formed method results from thefact that the effects of baseline drift are lessened when the algebraicsum of the two peaks is calculated.

In FIG. 1, band assignments were made by comparing the CD spectrum forthe total cholesterol, curve (a) in FIG. 1, with the spectrum for thesame sample after the selective precipitation of the low density lipidfractions with phosphotungstate-Mg, i.e., the shaded area in FIG. 1. The525 nm band maximum was calibrated using NBS cholesterol (SRM 911a).Calibration of the 390 nm maximum was done using secondary HDL-Ccalibrators supplied by Sigma Chemical Company.

As an example of carrying out one of the methods of the presentinvention and determining the amounts of cholesterol fractions in a testsample, there is provided Example 1:

EXAMPLE 1

(A) Calibration of the CD instrument

A 50 μl aliquot of a 5×10⁻³ M solution of (SRM 911a) cholesterol in ARgrade chloroform is placed in a vial of 10 mL total volume. 2.00 mL ofthe zinc chloride reagent are added and the mixture carefully shaken.1.00 mL of acetyl chloride is added with care, the mixture shaken, andthe vial capped and incubated at 67° C. for 8 minutes. The vial isremoved and cooled quickly under water. Chloroform (1.00 mL) is thenadded to increase the solution volume in the vial. Such an addition ofchloroform may be deleted if desired, if the CD analyzer willaccommodate a 2.00 mL sample volume, or alternatively, an appropriatesolvent substituted therefor. The solution is next transferred to a 1 cmpathlength cuvette and the CD spectrum run from 625 nm-325 nm. Thespectrum is corrected on a daily basis for the cell blank and theinstrument baseline by subtracting the spectrum for the reagent mixturealone.

(b) Calibration of the CD Spectra

The procedure in (a) is repeated for a number of solution concentrationschosen to coincide with the typical range of serum cholesterol levels inthe test sample. From the resultant calibration curve theproportionality constant relating the signal size at 525 nm to the(VLDL+LDL)-C level is 1.62 millidegrees per 100 mg/dL. The calibrationat 390 nm was done in the same way, but the pure cholesterol wassubstituted by Sigma HDL-C calibrators. The signal size to HDL-C levelat 390 nm is 2.08 millidegrees per 100 mg/dL.

(c) Cholesterol Determination in Clinical Test Samples by CD

The procedure in (a) is repeated for 50 μL aliquots of serum. Beforebeing transferred to the cuvette, the specimen is centrifuged at highspeed for 2 minutes. The (VLDL+LDL)-C fraction is calculated from themeasured signal height at 525 nm and the HDL-C fraction from the signalheight at 390 nm. Their sum give the total cholesterol in the specimen.Selective precipitation of the low density fraction in order to measurethe HDL-C fraction is not necessary in routine measurements. It ispossible therefore, to do a cholesterol-lipid profile with a volume aslittle as a finger stick, and get the best precision yet obtained in themeasurement of low density lipid fractions.

It should be noted that the reagents can be added in the order indicatedin (a) Calibration of the Instrument. However, they can also be addedsimultaneously as a premixed solution or they can be added in thereverse order, e.g. add the acetyl chloride first, followed by the ZnCl₂reagent. The latter mode of reagent addition had the unexpected effectof reducing the amount of precipitation in the test sample, therebygreatly reducing the scattering of incident light and therebysimplifying the subsequent measurement of absorption either by CD or byconventional spectroscopic absorption. An alternative is to use areagent comprising zinc acetate (in lieu of zinc chloride in glacialacetic acid) and acetyl chloride.

(d) Results of Exploratory Work

Cholesterol determinations were made on serum samples provided by twodifferent laboratories, which employ the commercial methods developed byAbbott Laboratories (Lab A) and DuPont (Lab B), respectively. Thecorrelations for total cholesterol levels are excellent, FIG. 2, andwell within the limits imposed by the LSP.

A good case for believing that this new method is an improvement overprior methods, is to compare the correlations for the three data setstreated independently. Plots of total cholesterol versus (VLDL+LDL)-Care linear in every case, but there is a bias of almost 50 mg/dL in theintercepts on the x-axis for both conventional methods (FIGS. 3(b) and3(c)) and zero correlation between the total and HDL-C data for thesesame data sets (FIGS. 4(b) and 4(c)). The Chugaev-CD data correlationsby comparison, are excellent with low correlation intercepts, FIGS. 3(a)and 4(a), and the correlation slopes indicate that, for these samplepopulations, the "average" percentages for the HDL-C and (VLDL+LDL)-Cfractions are 15% and 85%, respectively, which are in good agreementwith the values normally accepted as typical for human serumdistributions based upon ultracentrifugation data. Correlation slopesfor the previously known spectrophotometric absorption methods are bothone, which is not statistically possible, and which arises because thevirtually constant measured value of 50 mg/dL for HDL-C is subtractedfrom measured TC values to obtain the results for (VLDL+LDL)-C.

(e) Accuracy and Analysis Time

Since there are no commercial reference standards for either LDL-C orVLDL-C, the accuracy cannot be evaluated. However, the precision andrepeatability in the (VLDL+LDL)-C measurements are better than ±2%. Withthis level of precision, the confidence in one's ability to correlatethe changes in LDL-C in reduction therapy studies, which involved dietand/or exercise modifications, is meaningfully improved.

The approximate time for a single analysis by the Chugaev-CD method withCD detection is about 15 minutes. While this is long compared to thecommercial absorption methods used only for TC measurements, results forboth low and high density fractions are obtained simultaneously. Becauseof the stability of the color, the turn around time can be reducedconsiderably by incubating several samples at once. With greaterincident light intensities, sample path lengths can be reduced from 1 cmand the measurements can be automated.

Utilizing Chugaev reagents in procedures such as those provided above,several National Bureau of Standards SRM total cholesterol standardswere also examined. The three samples tested were listed in the NBScatalogue as (1951-1) (210.36±2.46 mg/dL total), (1951-2) (242.29±1.83mg/dL total), and (1951-3) (281.97±1.83 mg/dL total). According to theNBS Certificate of Analysis, the serum was donated by the CDC. Thefigures in parentheses are those measured at NBS and they compareextremely well with the CDC determinations using the modifiedAbell-Kendall method. The figures that we obtained from the Chugaevreaction, by adding the CD absorption values for the two fractions(HDL-C and (VLD+LDL)-C) were 206 mg/dL, 241.1 mg/dL, and 286.6 mg/dL,respectively. These results clearly evidence the effectiveness of thepresent inventive methods in determining cholesterol levels directly andprecisely.

In order to further evidence the effectiveness of the present inventiveCD methods in determining levels of cholesterol subfractions in a testsample, additional experimental data are provided in Table I.

                  TABLE I                                                         ______________________________________                                        Blood Fractions                                                               Patient  VL + LDL(Chug).sup.1                                                                        HDL(Chug.sup.2                                                                           HDL(enz).sup.3                              ______________________________________                                        A        126           31         [63]                                        B        165           28         [46]                                        C        220           33         --                                          D        237           34         [55]                                        E        199           29         32                                          F        188           39         36                                          G        249           36         43                                          H        199           34         25                                          I        144           28         [53]                                        J        216           46         52                                          K        190           38         35                                          L        211           41         --                                          M        239           39         --                                          N        190           39         [56]                                        O        220           50         --                                          P        174           46         46                                          Q        249           51         57                                           Q*      242           48         --                                          R        184           47         [60]                                        S        205           29         33                                          T        126           46         45                                          U        157           46         49                                           U*      163           41         --                                          V         94           31         [86]                                        W        293           38         --                                          X        239           47         [84]                                        Y        207           57         55                                          Sigma 400                                                                              340           51         --                                          Sigma H  230           52         --                                          ______________________________________                                         .sup.1 VL + LDL(Chug)  Cholesterol subfraction VLDLC + LDLC using Chugaev     reagents and taking CD absorption measurement at 575 nm.                      .sup.2 HDL(Chug)  Cholesterol subfraction HDLC obtained using Chugaev         reagents and taking algebraic sum of Cd absorption measurements at 390 an     574 nm.                                                                       .sup.3 HDL(enz)  subfraction HDLC obtained using the enzymatic method         designated by Lab(A) and Lab(B).                                              *Asterisk indicates test was performed on patient's serum using mixed         Chugaev reagents stored 4 week at 40° C.                               [ ] Brackets indicate HDL measurements which are substantially different      from HDL measurements using other methods.                               

Of the experimental results shown in Table I, it is noted that 12 out of20 values for each of the HDL-C(Chug) and HDL-C(enz) methods agree towithin 10 mg/dL. Such results clearly help to evidence the accuracy ofthe present methods.

B. Direct Detection Using Spectrophotometric Absorption Detection

The visible absorption spectrum for the colored product of the Chugaevreaction with cholesterol or serum cholesterol shows a strong maximumaround 518 nm, a minimum around 460 nm, with shoulders at wavelengthsbetween 460 nm and 365 nm showing a fairly weak absorption maximum (FIG.6). Additionally, the Chugaev reagent itself absorbs in the visiblerange and has a weak maximum in the 350-370 nm regions.

In order to ascertain the effectiveness of the present inventivespectrophotometric absorption detection methods, samples of separatedHDL-C, VLDL-C, and LDL-C lipoprotein fractions were obtained from SigmaChemical Company (Sigma; fractions separated by ultrafiltration) andfrom Oklahoma Medical Research Foundation (OMRF; subfractions separatedby ultra-centrifugation). The three subfractions were reacted separatelywith Chugaev reagents to give colored products which possess differentabsorption spectra in the visible range. Spectral correspondencesbetween the fractions from the two different sources were excellent forthe HDL-C and LDL-C samples. Correspondence for the VLDL-C subfractionswere also good. The spectral differences between each of thesubfractions is sufficient to enable the three cholesterol subfractionsto be qualitatively determined simultaneously in a single experimentwithout resorting to a selective precipitation step. For purposes ofthis example, absorption measurements are taken at 518, 450 and 420 nm.The serum spectrum of any test sample is an aggregate of the weightedcontribution from each subfraction.

The ability to calculate the amount of each cholesterol subtractionpresent in a test sample, is due to the inventors' initial postulationthat all three subtractions absorb at every wavelength analyzed, so thatthe general equation for total absorbance A_(T) of a serum test sampleis given by the equation:

    E.sub.HDL [HDL]+E.sub.VLDL [VLDL]+E.sub.LDL [LDL]=A.sub.T

In the above equation, the E coefficients denote the absorbances foreach of the subscripted fractions normalized in appropriate units ofabsorbance/(mg/dL), and the concentration terms [ ] are in mg/dL.Utilizing the above equation, and making the further assumption thateach subtraction has the same or a substantially similar absorptioncoefficient at 518 nm but, as exemplified by the different spectra, havedifferent absorption coefficients at the other wavelengths (in this case420 and 450 nm), it is possible to calculate the amount of eachsubtraction present in a test sample by taking an A_(T) measurement ateach of the three discrete wavelengths in the spectrum and solving theresulting 3·3 matrix equation. In order to do this, the individualvalues for the subscripted E coefficients have to be determined for thethree wavelengths selected. Relative values for the E coefficients atdifferent wavelengths were easily obtained within the spectrum of anyone of the subfractions. Relating these to the values of the equivalentwavelengths for the other fractions was more difficult, but was achievedand is disclosed herein. In this respect, spectral analysis of about 90serum samples showed that a direct linear correlation existed betweenthe A_(T) values measured at 518 nm and the value for total cholesterol(TC) measured in a completely independent study that utilized aconventional method (data from Roche Biomedical). Based on this linearcorrelation, the inventor presupposed that a normalized value expressedas A_(T) /TC should be constant from sample to sample.

In order to correlate E values between the spectra for the differentsubtractions, the inventor postulated that the A_(T) /TC (or E) valuesat 518 nm are the same for all three subfractions, as noted above, andthat the remaining six E coefficients for the three subtractions can becalculated for the remaining two wavelengths using simple proportions(e.g., E_(LDL)(420) =A_(T)(420) /A_(T)(518) ×E_(LDL)(518).

The amount of the above three lipofractions calculated from 60 serumsamples utilizing the above technique and 3·3 matrixes provided resultsin excellent agreement with numbers obtained for the same test samplesutilizing a conventional reaction procedure.

The specific test procedure utilized with the 60 samples that gaveexcellent agreement was as follows. After reagents were added themixtures were allowed to incubate for 8 minutes at 67° C., thereaftercooled in a waterbath, centrifuged, transferred to a one cm cuvette, anda conventional absorption spectrum run from 700 nm-400 nm. Absorptionmeasurements were taken at 518, 450 and 420 nm, after appropriatecorrections for the cell blank and the instrument base line were made.It is not necessary to run the entire spectrum, since absorbancemeasurements are only needed at the prescribed wavelengths. The nine Evalues for the wavelengths 518, 450 and 420 nm, respectively, obtainedunder the above described particular experimental conditions in units ofabsorbance, dL/gram were as follows: 3.05, 1.97 and 2.52 for HDL-C;3.05, 1.35 and 2.41 for VLDL-C; and 3.05, 1.31 and 1.34 for LDL-C.

With Chugaev reagents problems may arise in the use of absorptionspectrophotometry which do not exist when CD methods are used.Specifically, whenever an absorption detection spectrophotometric methodis utilized with Chugaev reagents precipitation may create problems. Inorder to fully minimize such problems, the Chugaev reagent should bemodified such that either the ratio of acetyl chloride to zinc reagentis from 100:1 to 4:1 or zinc acetate is substituted for zincchloride/acetic acid. The final zinc concentration should be between0.03 and 0.22 molar. This is a composition much different from thereagent described in the literature.

It is noted that excellent absorption spectra have been obtained forvolumes of serum as little as 2 μl. Likewise, excellent spectra wereobtained for various acetyl chloride to serum ratios over the generalbroad range of 100:1 to 20:1 at constant zinc concentrations, and foracetyl chloride to zinc reagent ratios from 100:1 to 4:1 at constantserum amounts. Measurements have also been made with test samples,wherein the total reaction volume was as little as 0.15 mL and incuvettes having a pathlength as short as 1 mm. Moreover, incubationtimes as little as two minutes have been achieved with the smaller totalvolumes, and it is fully envisioned that under conditions where serumconcentrations are relatively high, that lower incubation temperaturemay be utilized. Furthermore, with appropriate miniaturization,centrifugation may be eliminated.

In addition to the Chugaev reagent system described above, it is notedthat ACS reagent grade zinc acetate dihydrate readily dissolves inacetyl chloride to a concentration that is similar to the final zinc ionconcentration when added as the chloride in glacial acetic acid. Zincacetate in acetyl chloride therefore can work, if desired, as a singlereagent system. For example, if one mL of such a reagent system is addedto 10 μL of a test serum, there is obtained a reddish-orange productafter the usual incubation conditions. The maxima and minima in thespectra are at the same wavelengths but the ratios of the heights ofthese bands are different from those seen with the zinc chloridereagents with the unexpected finding of a greater difference between theabsorbances at 420 and 450 nm. Consequently, new E coefficients wouldneed to be calculated for a 3·3 matrix if the zinc acetate were usedrather than zinc chloride in acetic acid. The greater difference atthose wavelengths means better precision in the values obtained for thesubfractions. Indeed, the coefficients are highly dependent on thecomposition of the reagent and must be recalculated if the amounts ofany of the reagent components are changed. However, such a calculationis in line with those earlier described, and clearly within the skill ofthose of ordinary skill in the art, based on the present disclosure.

A broad range of alternative reaction conditions to those reactionconditions discussed above, will produce reddish-orange colored productswhich have spectra that are similar, but not always equivalent to theabsorbance spectrum of the species produced under the exact conditionsutilized herein (as described above). Even so, when those skilled in theart utilize such alternative reaction conditions in combination with the3·3 matrix strategy provided herein, and calculate the amounts of eachsubfraction present, they are practicing the inventor's presentlydisclosed methods. This is true, even though the nine E coefficientsutilized may have to be revised after a recalibration of the spectra forstandards of each of the subfractions (e.g., Sigma or OMRF providedsubfractions) based on the exact reaction conditions employed. As such,it is envisioned that the present spectrophotometric methods clearlycover all such possible reagent mixtures and reagent ratios, so long asa colored reaction product is formed with the cholesterol subfractions,and the amounts of each subfraction are then determined in a manner asdescribed above.

The above-described spectrophotometric absorption methods offer anopportunity for simultaneous, on-line detection of cholesterol andcholesterol subfractions in clinical samples. The use ofspectrophotometric absorption methods in accordance with the presentinvention also permits much greater sensitivity than the CD methodsherein disclosed allow for, since only a very small portion of theincident light can be used for CD signal generation. As such, thespectrophotometric absorption methods herein disclosed also permit theuse of smaller volumes of sample, thereby reducing possibleinterferences caused by other materials and the total amount ofprecipitates formed by the reaction. Conversely, however, thesereactions are more susceptible than CD to interferences from pigmentsreleased by hemolysis of the blood samples. Finally, it is important tonote that, as with the CD studies mentioned above, addition of theacetyl chloride to the sample first, followed by addition to the ZnCl₂/acetic acid reagents reduces even further the interferences caused byprecipitation in a clinical sample. Indeed it is possible to carry outspectrophotometric absorbance reactions in the present inventive methodsusing whole blood samples.

In the following Table II, there is provided comparative data obtainedwith test samples using both the spectrophotometric absorption/Chugaevmethod disclosed herein and an enzymatic method for the cholesterolsubfractions shown. As may be seen upon review of Table II, excellentresults were obtained using the Chugaev reagents/spectrophotometricabsorption method herein described (as verified by comparing withresults obtained on the same test samples using the enzymatic method).

                  TABLE II                                                        ______________________________________                                        Subfractions from Enzymatic and Chugaev Methods                               Test                                                                          Subject                                                                              Test      VLDL-C    LDL-C  HDL-C  TC                                   ______________________________________                                        1      enzymatic 32        155    28     216                                         Chugaev   29        155    35     222                                  2      enzymatic 46        178    37     262                                         Chugaev   50        173    42     264                                  3      enzymatic 37        110    36     183                                         Chugaev   48        103    31     182                                  4      enzymatic 34        133    46     214                                         Chugaev   38        139    36     213                                  5      enzymatic 42        155    45     242                                         Chugaev   44        161    41     246                                  6      enzymatic 62        113    26     202                                         Chugaev   65        101    35     203                                  ______________________________________                                    

Based on the above considerations, there is provided herein a novelspectrophotometric absorption detection method, wherein cholesterolsubfractions in clinical samples are reacted with either Chugaevreagents or the acyl compound and perchlorate reagent system as definedhereinabove, so that a direct measurement of the cholesterolsubfractions can be made. The measurements can be made either as a fullspectrum over the range of about 150-700 nm or at 3 selectedwavelengths, in this case about 420 nm, 450 nm and 518 nm.

The major procedural difference between the absorption and the CD methodrelates to the standards used. While cholesterol itself can be used as astandard for the CD reactions, clinical standards for TC and cholesterolsubfractions obtained from the CDC, CAP or a commercial source must beused to calibrate the absorption spectrometer.

Further to the above disclosed spectrophotometric methods, given theavailability of "pure" samples of VLDL-C, LDL-C and HDL-C, amathematical algorithm can be prepared, if so desired, which enables oneto add the individual subfractions' spectrophotometric absorptionspectra in a weighted fashion for each subfraction. In such a manner thetotal absorption spectrum for the test sample is obtained. Utilizingsuch a method would be analogous to measuring the spectrophotometricabsorption of a colored reaction product at an infinite number ofpoints, instead of just at three or more distinct points as describedabove.

As an example of practicing the material of the present inventionwhereby a spectrophotometrically active product of cholesterol in a testsample is formed by contact with an acyl compound and a perchlorate ashereinbefore defined, Example 2 is provided.

EXAMPLE 2

Experimental Conditions

The reagent consisted of an 0.5 M solution of zinc perchlorate sixhydrate [Zn(ClO₄)₂ ·6H₂ O)] in 98% acetyl chloride. After mixing, thesolution was centrifuged to remove suspended materials, most probablyundissolved ZnO, which is reported by the manufacturer to be a possibleimpurity. The reagent was stable when stored in a tightly sealedamber-glass container.

A 20 μL aliquot of serum was placed in a glass or polypropylene vial and2 mL of reagent was carefully added and the mixture was shakenthoroughly. Transfer proteins were precipitated on the addition of thereagent and were quickly and easily removed either by centrifugation orfiltration. The supernate was transferred to a sealed, 1 cm pathlength,spectrophotometric cuvette and allowed to stand at room temperature for15 minutes at which time the absorption spectrum was measured from750-380 nm. The instrument used was a diode array spectrophotometer, butthe necessary data for the lipid profile analysis was obtained usingonly four wavelengths, one of which (700 nm) was used for the samplebaseline correction. The other wavelengths were 410 nm, 456 nm and 518nm.

The values for total cholesterol in a sample was determined from theabsorbance measured at the major maximum, which under the aboveexperimental conditions occurred at 518 nm. The constant that allowedtotal cholesterol to be calculated was obtained from the slope of thelinear correlation between measured absorbance A_(T) (518) and the totalcholesterol measured by the commercially available enzymatic method. Themathematical procedure to calculate the concentration of the three majorfractions, VLDL-C, LDL-C and HDL-C was the same as hereinbeforedescribed, i.e., by solving the 3×3 matrix that consists of three linearBeer's Law equations given by the sum of the absorbance for eachfraction:

    A.sub.T(i) =A.sub.VLDL-C(i) +A.sub.LDL-C(i) +A.sub.HDL-C(i)

wherein i=1-3.

Each A term for a fraction consisted of a product of the concentrationof the fraction times a coefficient E: e.g., A_(LDL-C)(i) =E_(LDL-C)(i)·[LDL-C]. E values are usually defined as molar absorbances but for thisapplication the units were converted to mg/dL which is standard forreporting lipid data. The solution required that the nine E coefficients(one for each fraction at three wavelengths) be determined. This couldnot be done exactly because there are no known. samples of referencestandards available for the lipid fractions. Therefore, they werearrived at empirically. This was done by comparing lipid panels for alarge pool of serum samples that had been measured by the commerciallyavailable enzymatic method and by the method of the present invention.The coefficients were systematically adjusted as the size of the poolwas enlarged to give the best statistical fit among data for all thefractions.

The results for the lipid profile analysis is presented below in TableIII. Enzymatic refers to the independent results measured by RocheBiomedical, Kansas City; spectrophotometric refers to the results fromthe Chugaev reaction described herein; and kinetic refers to the resultsfrom the use of zinc perchlorate hexahydrate and acetyl chloride.

                  TABLE III                                                       ______________________________________                                        Comparisons of Lipid Panels by Three Methods                                  A              B       C        D     E                                       ______________________________________                                         1   Method                                                                    2                 Total   VLDL   LDL   HDL                                    3   Sample 1                                                                  4   enzymatic     160     20     100   40                                     5   spectrophotometric                                                                          170     25     100   45                                     6   kinetic       150     12      90   48                                     7                                                                             8   Sample 2                                                                  9   enzymatic     179     13     124   41                                    10   spectrophotometric                                                                          181     13     121   47                                    11   kinetic       165      5     113   47                                    12                                                                            13   Sample 3                                                                 14   enzymatic     215     39     141   35                                    15   spectrophotometric                                                                          229     36     131   62                                    16   kinetic       208     23     126   58                                    17                                                                            18   Sample 4                                                                 19   enzymatic     204     26     141   36                                    20   spectrophotometric                                                                          213     24     132   57                                    21   kinetic       195     20     125   49                                    22                                                                            23   Sample 5                                                                 24   enzymatic     195     68      98   28                                    25   spectrophotometric                                                                          217     52     106   59                                    26   kinetic       202     46     104   52                                    27                                                                            28   Sample 6                                                                 29   enzymatic     196     26     129   41                                    30   spectrophotometric                                                                          200     20     131   49                                    31   kinetic       198     23     126   49                                    32                                                                            33   Sample 7                                                                 34   enzymatic     233     39     151   42                                    35   spectrophotometric                                                                          243     28     153   62                                    36   kinetic       229     28     148   54                                    37                                                                            38   Sample 8                                                                 39   enzymatic     132     36      65   31                                    40   spectrophotometric                                                                          135     36      60   39                                    41   kinetic       119     27      61   30                                    42                                                                            43                                                                            44   Sample 9                                                                 45   enzymatic     232     19     160   50                                    46   spectrophotometric                                                                          231     15     156   60                                    47   kinetic       231     18     164   49                                    48                                                                            49   Sample 10                                                                50   enzymatic     168     14     109   44                                    51   spectrophotometric                                                                          177     21     108   48                                    52   kinetic       153     20     100   34                                    53                                                                            54   Sample 11                                                                55   enzymatic     170     46      95   28                                    56   spectrophotometric                                                                          184     56      85   43                                    57   kinetic       178     32     100   45                                    58                                                                            59   Sample 12                                                                60   enzymatic     186     10     113   62                                    61   spectrophotometric                                                                          193     21     119   53                                    62   kinetic       170     10     108   50                                    63                                                                            64   Sample 13                                                                65   enzymatic     201     30     111   59                                    66   spectrophotometric                                                                          215     49     104   62                                    67   kinetic       208     24     128   58                                    68                                                                            69   Sample 14                                                                70   enzymatic     180     26     113   40                                    71   spectrophotometric                                                                          186     36      97   53                                    72   kinetic       186     24     116   46                                    73                                                                            74                                                                            75   Sample 15                                                                76   enzymatic     186     17     127   41                                    77   spectrophotometric                                                                          194     28     111   55                                    78   kinetic       182     21     120   44                                    79                                                                            80   Sample 16                                                                81   enzymatic     175     15     106   54                                    82   spectrophotometric                                                                          186     26     111   49                                    83   kinetic       180     24     119   37                                    84                                                                            85                                                                            86                                                                            87   Sample 17                                                                88   enzymatic     173      9     113   50                                    89   spectrophotometric                                                                          172     22     102   48                                    90   kinetic       176     18     118   39                                    ______________________________________                                    

EXAMPLE 3

In this example, zinc perchlorate hexahydrate and acetyl chloride werecontacted with cholesterol in a test sample following substantially theprocedure described in Example 2; however, the calculation ofcholesterol in this example was by way of multivariate regressionanalysis instead of a 3×3 matrix used in Example 2.

Incubation was performed for 15 minutes at ambient temperature. Proteinthat precipitated on addition of a 2 mL aliquot of zinc perchloratehexahydrate/acetylchloride reagent to 20 μL of serum was removed bycentrifugation or by filtration. The batching of samples reduced thetime per test to approximately 5 minutes.

Absorbance Measurements

Full spectral (350-750 nm) absorbance data for a sample with apathlength of 1 cm were collected on a Hewlett Packard 8452A diode arrayspectrophotometer; accumulation time was 5 seconds. With the speed andconvenience of diode array detection technology, the wavelength rangewas wider than that used for the method employing Chugaev reagents.

Computational

Since pure forms of cholesterol lipid fractions are not available,neither are the spectra for the individual products of the colorreaction. Therefore a mathematical model was developed to resolve thewhole spectrum into the contributions from the parts.

(a) 3×3 Matrix Solution

In Example 2, absorbances, A.sub.(i), were measured at three principalwavelengths and the lipid profiles were calculated by solving a set ofthree simultaneous equations of the form:

    A.sub.(i) =E.sub.VLDL(i) [VLDL]d+E.sub.LDL(i ) [LDL]d+E.sub.HDL(i) [HDL]d

where d was the sample pathlength. The nine E coefficients wereevaluated in an empirical manner.

(b) Multivariate Regression Analysis (MVRA)

Lipid profile and TC results in this example were determined using MVRAtechniques to interpret the full absorbance spectrum, which eliminatedsimplifying assumptions and investigator bias. The MVRA algorithms thatwere applied were Partial Least Squares 2 (PLS2) and Principal ComponentAnalysis (PCA) using software for spectroscopic analysis available inthe commercial package UNSCRAMBLER II (CAMO A/S, Trondheim, Norway).Spectral resolution in the spectrophotometer was 2 nm so a full spectrumconsisted of 200 data points, which represented an enormous increase inthe number of degrees of freedom compared to the simpler analysis ofExample 2.

Training Set

An analytical model was prepared by compiling a training set thatconsisted of 35 serum samples for which the lipid profiles had beenmeasured by commercially available enzymatic techniques; VLDL-C here wastaken to be 0.2×TG and numbers of LDL-C were calculated using theFriedewald formula. Ranges in values were as wide as could be accessed,namely; 8-80 mg/dL for VLDL-C; 85-222 mg/dL for LDL-C; and 20-80 mg/dLfor HDL-C. Using both the PLS2 and PCA algorithms, the optimum fit tothe spectral data for these 35 samples was obtained using three factors.The percent residual variance that was observed was about 25% with onlythe first factor and less than 0.05% for all three. No corrections weremade for noise or background and no weighting corrections wereintroduced.

As a test to determine if full spectral analysis was really necessary,the MVRA subroutines were used to identify the optimum wavelengths,i.e., those that are most sensitive to variations in the amounts of eachfraction. Alternative models were prepared using reduced data setslimited to 100, 30, 14, 6 and 4 wavelengths respectively. Littledifference was seen in the percent residual variance through the modelwith 6 points, and 4 could be used with little loss in the quality ofthe fit. For purposes of the present example, absorbances were measuredat six wavelengths.

Sample Predictions

The above-described analytical model was used to predict lipid profilesfor several hundred samples which were about equally split betweenregular TG and high TG levels. The major source of full lipid profiledata for comparisons between methods was Roche Biomedical, Kansas City,which utilized commercially available enzymatic techniques. The majorsource for HTG samples was the Stillwater Medical Center. For thelatter, only TC and TG levels were reported.

The formation of the spectrophotometrically active product was performedwith the HTG samples in precisely the same way and it was assumed thatthe same model could be extrapolated to include them. Four of the thirtyfive in the original training set were HTG samples. For these four, TGlevels were slightly above 400 mg/dL; HDL values were measured, andlipid profiles calculated.

In this treatment, values for the fractions were determined directly andTC was calculated from their sum. Thus, unlike the enzymatic methods, TCwas not measured experimentally.

Absorbance Spectra

A comparison of spectra for the colored products from one regular andtwo HTG serum samples is shown in FIG. 10. All three had a TC of about186 mg/dL, as measured enzymatically. The critical part of the spectrumfor discrimination among the lipid fractions was in the range of about360 to about 480 nm.

Training Set

As seen in FIG. 11, very good linear correlations for VLDL-C, LDL-C andTC between methods was obtained for the training set. As seen in FIG.12, the linear correlation for HDL-C was not nearly as good, but itshowed an improvement over the Chugaev-reagent method. The result from apaired t-test suggested that there was a correlation, and furtherevidence to support such a correlation was given by the shape of thebivariate ellipse which, as shown in FIG. 12, was drawn with aconfidence region of 65%, which meant that for a normal distribution inX and Y, 65% would lie within the ellipse.

Predictions

Regular Samples

As shown in FIG. 13(a), the between-methods linear correlation for TCwas excellent. The range was from about 58 to about 500 mg/dL and thefigure included data for HTG as well as regular samples. This wassignificant because TC is measured in one method and calculated in theother, which validated the MVRA models used for the spectralinterpretation.

As shown in FIG. 13(b), the between-methods linear correlations werealso very strong for the LDL-C fraction, which suggested that themeasurement of LDL-C by the direct method of the present invention wasreliable. As shown in FIG. 13(c), the correlation of HDL-C was onlyslightly worse than it was for the training set. The VLDL-C correlationfor samples with TG less than about 400 mg/dL was good, as shown in FIG.14(a).

HTG Samples

Absorbances in the range of about 360 to about 430 nm increaseddramatically with increasing TG. It was significant that the increase inthe maximum absorbance at 360 nm was non-linear with the amount of TG,as shown in FIG. 10. A linear dependence would only be expected if theband could be assigned entirely to VLDL-C absorption and theapproximation VLDL-C=0.2 TG were true for HTG samples, which it was not.Meaningful results for lipid profiles were obtained for HTG sampleswhere the TG level was as high as 2000 mg/dL. Between 400 and 1000mg/dL, the VLDL-C/TG ratio was seen to decrease from 0.2 to 0.12 whichis manifested in the curvature observed in the plots of VLDL-C (spec)vs. TG, as shown in FIG. 14(b). This was consistent with the fact thatthe Friedewald equation, with VLDL-C=0.2×TG, fails at high TG levels.

An inverse correlation between HDL-C and TG has sometimes been alludedto. As long as the routine measurement of VLDL-C was limited to 0.2×TGand TG values were less than TG=400 mg/dL, the correlation had not beenobvious. As shown in FIG. 15, adding VLDL-C data for HTG samplesemphasized the relationship.

C. Direct Detection Using Fluorescence and Derivative AbsorptionSpectrophotometric Methods

The products of the reaction of cholesterol with the Chugaev reagentsare fluorescent. Moreover, fluorescence spectra for the threelipoprotein subfractions VLDL-C, LDL-C and HDL-C are different from eachother and from the spectrum for a serum sample (see FIGS. 7(a), 7(b) and7(c)). The mathematical analysis of fluorescence data, wherein onecalculates the amounts of each of the subfractions present in a serumtest sample is entirely equivalent to that described above for theconventional absorbance detection spectrophotometry. All that isrequired to initiate the calculation are the nine fluorescencecoefficients for whatever three wavelengths are selected in thefluorescence spectrum for serum. In this respect, wavelengths differentfrom those utilized in conventional absorption spectrophotometry areneeded, since the maximum and minimum wavelengths for fluorescence occurat longer wavelengths.

Similarly, derivative absorption spectrophotometry may also be utilizedto calculate the amount of a cholesterol subfraction present in the testserum sample. For example, first and second derivatives of absorbancespectra can be utilized for analytical measurements. Copies ofderivative absorbance spectra for each of the three lipoproteinsubtractions are shown in FIG. 8. FIG. 8(a) shows the first and secondderivative for VLDL-C; FIG. 8(b) shows the first and second derivativefor LDL-C; and FIG. 8(c) shows the first and second derivative forHDL-C. In each of FIGS. 8(a)-8(c), the solid line denotes a firstderivative to the absorbance spectrum and the dotted line denotes thesecond derivative of the absorbance spectra. Each of the subtractionsutilized to obtain the graphs 8(a)-8(c) were obtained from SigmaChemical Company. When utilizing derivative absorptionspectrophotometry, subtle differences exist between the spectra for eachof the fractions. Again, the mathematical analysis is completelyanalogous to that discussed above for absorbance detection andfluorescence spectrophotometries. However, three new wavelengths wouldneed to be chosen. Signal intensities at the band maxima are much betterseparated than with other methods and precision may therefore beincreased. In two measurements on serum samples (see FIGS. 9(a) and9(b)) it was determined that the peak to peak heights for the two majorbands were directly proportional to TC. Data collection utilizingderivative spectrophotometry requires the use of a full spectrumanalysis.

INVENTIVE APPARATUS

Upon review of the above methods section, it can be easily ascertainedthat the present inventive methods have many advantageous attributeswhen compared with presently known methods for determining cholesterollevels in test samples. However, the present invention also encompassesnovel instruments, which allow those skilled in the art to practice thepresent inventive methods. Such inventive instruments are outlined above(see Section entitled "Summary of the Invention").

A spectrophotometric instrument encompassed hereby should be equippedwith 1 or more spectrophotometric absorption detectors capable ofmeasuring the absorption of the colored products of the Chugaev reagentover a range of from about 360-700 nm, or at discrete points thereinsuch as about 518 nm, 450 nm and 420 nm. If automated, it should alsohave the capability of adding the reagents to separate sample containersfor analysis or to sequentially add the components of the reagents tominimize problems due to precipitation of proteins. Finally, any suchabsorption spectrophotometer, manual or automatic, should preferablyhave the means to determine the levels of each subfraction present in aserum test sample by a calculation or computation from the absorptionvalues obtained. Specifically, the instrument should have the ability tocompute the results of the 3·3 matrix, with nine pre-programmedconstants, to establish the levels of VLDL-C, LDL-C and HDL-C present,and to use these values to compute the TC present in the sample, oralternatively, should be equipped with the ability to employmultivariate regression analysis to establish these levels.

It should be noted that the 3·3 matrix represents the minimum possibleto measure the three subfractions. It is possible that finer analysis ofthe spectrum produced by the reagent will indicate that constants atother specific wavelengths will provide useful information, e.g., aboutspecific molecular entities within the various subfractions. In thatcase, the instrument should be construed to analyze matrixes larger than3·3.

EXAMPLE 4

This example describes the use of modifier to control the rate ofreaction using acyl compound and perchlorate.

Modification of Reagent to Manage Reaction Rates

The rate of the color reaction and the reagent using acetyl chloride andzinc perchlorate hexahydrate can be increased or decreased by using amodifier in the following manner:

(a) a rate decrease by a factor of two or more was observed when eitherwater, or glacial acetic acid, or chloroform at a level greater than 10%v/v was added to acetyl chloride. Water must be added with great carebecause of the heat of mixing. At water levels greater than 20% a bluecolored solution was produced due to reaction with protein. At 50%chloroform the reagents were immiscible. The rate was controlled byselecting the appropriate mole fraction for the added solvent.Retardation of the reaction at 25° C. by the addition of any of thesesolvents can be used to advantage if a temperature of 37° C. ispreferred for the reaction.

(b) a rate increase was observed when strong acid (HCl or HClO₄) wasadded in the amount of 1.2% v/v. Again great care must be used whenadding the aqueous acids. The spectrum after 10 minutes was the same asthat for the unmodified reagent after 15 minutes, but the spectrum forthe acid mixture changes considerably over the next 5 five minutes somore careful control of the condition would be necessary.

Zinc perchlorate hexahydrate dissolved easily in acetyl chloride and thereagent has a relatively long shelf life in a sealed container underambient conditions. The shelf life was extended when the reagent wasstored in the refrigerator. With some commercial products a slightamount of insoluble material is left. This was readily removed by slowspeed centrifugation and the data for this reagent compared exactly withdata where the zinc perchlorate completely dissolved. For measurementsat a temperature of 25° C. the procedure called for the thorough mixingof 2 mL of the modified single reagent with a 20-50 μL aliquot of serum,centrifuging (or filtering) the mixture to remove precipitated proteins,and measurement. The visible spectrum of the product changed with timeand the reaction was approximately 95-98% complete after 15 minutes atwhich time the absorbance spectrum was measured from 700-400 nm againsta reagent blank. The spectrum for the colored product after 15 minutesis analogous to that observed for the zinc chloride (modified Chugaev)reagent with a slight blue shift in the wavelengths of the minimum andsecond maximum.

Calculations of TC and its distribution among the three majorlipoproteins were done in exactly the same way as before, e.g., a 3×3matrix of linear Beer's Law equations are set up for three distinctwavelengths, at 518, 456 and 410 nm. Reagent blank and instrumentbaseline were measured at 700 nm. Absorbance coefficients weredetermined empirically as before, by first assuming that all threefractions had the same molar absorbance at 518 nm and subsequentlyratio-ing respective absorptions at 518 nm. For VLDL-C the currentadjusted coefficients are: 2.70, 2.90; for LDL-C they are: 2.70, 1.15and 1.20; and for HDL-C they are: 2.70, 1.25 and 2.30.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A chemical reagent which comprises an acylcompound having the formula: ##STR3## wherein R₁ is halogen, R isselected from the group consisting of lower alkyl; and a perchlorateeffective to form a spectrophotometrically active product withcholesterol, said percholorate selected from the group consisting ofbarium perchlorate and perchloric acid.
 2. The reagent of claim 1wherein said spectrophotometrically active product is capable ofdetection by circular dichroism, absorption spectrophotometry,fluorescence spectrophotometry or derivative absorptionspectrophotometry.
 3. The reagent of claim 1 wherein said perchlorate ispresent in said reagent at a concentration of about 0.3-0.7 molar inabout 90-100% acyl compound.
 4. The reagent of claim 3 wherein saidperchlorate is present in said reagent at a concentration of about0.4-0.6 molar in about 95-99% acyl compound.
 5. The reagent of claim 4wherein said perchlorate is present in said reagent at a concentrationof about 0.5 molar in about 98% acyl compound.
 6. The reagent of claim 1further comprising a modifier to control the rate of reaction at whichsaid spectrophotometrically active product is formed.
 7. The reagent ofclaim 6 wherein said rate is decreased and said modifier is water,glacial acetic acid, chloroform or mixtures thereof.
 8. The reagent ofclaim 7 wherein said modifier is present in said reagent at aconcentration of greater than about 10% v/v based upon said acylcompound.
 9. A chemical reagent which comprises an acyl compound havingthe formula: ##STR4## wherein R₁ is halogen, R is lower alkyl; aperchlorate effective to form a spectrophotometrically active productwith cholesterol, said perchlorate selected from the group consisting ofzinc perchlorate, barium perchlorate and perchloric acid; and a modifierto increase the rate of reaction at which said spectrophotometricallyactive product is formed, wherein said modifier is HCl, HClO₄ ormixtures thereof.
 10. The reagent of claim 9 wherein said modifier ispresent at a concentration of about 1-2% v/v based upon said acylcompound.
 11. A chemical reagent which comprises an acyl compound havingthe formula: ##STR5## wherein R₁ is halogen, R is lower alkyl; aperchlorate effective to form a spectrophotometrically active productwith cholesterol, wherein said perchlorate is zinc perchlorate; and amodifier to control the rate of reaction at which saidspectrophotometrically active product is formed.
 12. The reagent ofclaim 11 wherein said zinc perchlorate is a zinc perchlorate hydrate.13. The reagent of claim 12 wherein said zinc perchorate is zincperchlorate hexahydrate and said acyl compound is acetyl chloride. 14.The reagent of claim 11 wherein said rate is decreased and said modifieris water, glacial acetic acid, chloroform or mixtures thereof.
 15. Thereagent of claim 14 wherein said modifier is present in said reagent ata concentration of greater than about 10% v/v based upon said acylcompound.
 16. The reagent of claim 11 wherein said rate is increased andsaid modifier is HCl, HClO₄ or mixtures thereof.
 17. The reagent ofclaim 16 wherein said modifier is present at a concentration of about1-2% v/v based upon said acyl compound.